Herpes simplex virus (HSV) infection of epithelial cells often leads to spread of virus within the epithelium and to neurons for the establishment of a latent infection. Reactivation results in spread of virus from neurons back to epithelial cells. The virus can spread destructively in an epithelium, often in the presence of neutralizing antibody, resulting in the formation of a characteristic lesion. HSV spreads from an infected cell to an uninfected cell in three ways. First, extracellular virus binds cell-surface receptors on the adjacent cell and enters by fusion of the viral and cellular membranes (viral entry). Second, viral glycoproteins on the surface of the infected cell induce fusion with adjacent uninfected cells resulting in giant-multinucleated cells called syncytia (cell fusion). The third form of viral spread occurs between contiguous cells in the presence of neutralizing antibody and absence of syncytium formation (cell-cell spread). A cell-surface gD receptor, such as nectin-1, is required for all three forms of spread. The long term objective of these studies is to characterize the mechanisms of all three forms of nectin-1-dependent viral spread. We hypothesize that the structural features of nectin-1 that are critical for these three activities (and therefore the mechanisms) may differ, particularly for cell-cell spread. To test this hypothesis, we will identify domains of nectin-1 that are important for cell fusion and cell-cell spread. We will further characterize the mechanism of cell-cell spread by examining the functional requirements of adherens-junction proteins for efficient cell-cell spread in epithelial cells. The results of these studies, when compared and contrasted with previous work in viral entry, will elucidate nectin-1-mediated events at the cell membrane during viral spread. These results are important for the characterization of viral spread in an epithelium and may also be important for the characterization of spread of virus to neurons and interneuronal spread. Results from these studies will contribute to the understanding of viral spread in human and animal herpesviruses.